1. Filed of Invention
The present invention relates to the field of molecular biology. More specifically, the invention relates to methods, compositions, kits and apparatus for detection, identification and quantification of biomolecules. In certain embodiments of the invention, the biomolecules may be a nucleoside, nucleotide, oligonucleotide, polynucleotide, nucleic acid and other biological agents known to those skilled in the art. In particular embodiments of the invention, the methods involve use of enzymatic luminescence reaction and optical detection for quantitation the number of target molecule in biological samples.
2. Description of Related Art
Recognition and quantitation of level of DNA and RNA (gene expression) in biological samples is required in many fields of life science research, drug discovery, clinical diagnosis, and environmental analysis. While microarrays emerged as a dominant tool for large-scale DNA analysis and gene expression research, the alternative non-array gene expression technologies can be superior to microarrays in accuracy and sensitivity. Non-array techniques include quantitative Polymerase Chain Reaction (RT-qPCR), Serial Analysis of Gene Expression (SAGE), Northern blots, differential display, and Massively Parallel Signature Sequencing (MPSS). Now considering quantitation of gene expression as example, it is appreciated that some embodiments of this invention can be applied for recognition and quantitation of biomolecules including a nucleoside, nucleotide, oligonucleotide, polynucleotide, nucleic acid, peptide, polypeptide, protein, carbohydrate, polysaccharide, glycoprotein, lipid, hormone, growth factor, cytokine, receptor, antigen, allergen, antibody, substrate, metabolite, cofactor, inhibitor, drug, pharmaceutical, nutrient, toxin, poison, explosive, pesticide, chemical warfare agent, biowarfare agent, biohazardous agent, infectious agent, prion, radioisotope, vitamin, heterocyclic aromatic compound, carcinogen, mutagen, narcotic, amphetamine, barbiturate, hallucinogen, waste product, contaminant, and other biological agents known to one skilled in the art.
Microarrays have been increasingly applied as a multiplexed tool for monitoring expression levels of thousands of genes in samples under various conditions. Microarrays have been undeniably successful in many applications. Yet, data quality is a major concern of microarray-based studies. It is clear that the validation of results from microarray experiments by other methods is essential. Indeed, multiple factors can contribute to the errors, including variations in microarray manufacturing, cDNA generation and labeling, hybridization, and instrumental errors during image acquisition and quantification. Quantitative RT-qPCR and Northern blot are two most commonly used follow-up techniques for validation of microarray results, as these techniques have higher sensitivity and accuracy.
The real time quantitative PCR (RT-qPCR) is often considered to be the gold standard and the method of choice for mRNA quantification. The method utilizes a pair of synthetic oligonucleotides, or primers, each hybridizing to unique sites on the two strands of a double-stranded DNA (dsDNA) target, with the pair spanning a region that will be exponentially reproduced. The hybridized primers act as substrates for DNA polymerase, which create a complementary strand via sequential addition of deoxynucleotides to hybridized primers. An intercalating fluorogenic agent (SYBR green) or dye/quencher reporter probes are employed for monitoring the progression of PCR amplification. The amount of target RNA is measured by determining the number of amplification cycles, Ct, required to produce amount of PCR products that exceed a certain threshold level of detection.
Several factors have contributed to the transformation of RT-qPCR technology into a mainstream research tool: (i) as a homogeneous assay it avoids the need for post-PCR processing; (ii) a wide (>107-fold) dynamic range allows straightforward comparison between RNAs that differ widely in their abundance; and (iii) the assay realizes the inherent quantitative potential of the PCR, as well as its qualitative uses.
Yet, RT-qPCR is not pitfall-free. Some of the problems have their roots in quantitative nature of this technology and may result from the close association between quantification and amplification efficiency. Often the nature and extent of the unreliability of quantitative RT-PCR data is still not widely appreciated or acknowledged. One example is the threshold cycle Ct, which records the cycle when sample fluorescence exceeds a chosen threshold of the background fluorescence. The Ct is used for quantifying target copy number, yet its value is subjective and can be altered at will making it difficult to compare results from different platforms and at various experimental conditions. Another important issue is the reverse transcription step of RT-qPCR process. What is often pursued as a simple and small step of converting RNA into a cDNA template is an important contributor to the variability and lack of reproducibility frequently observed in RT-qPCR experiments. This is especially the case when cDNA priming in real-time RT-qPCR assays is carried out using random primers or oligo(T). The random primers create problems if the mRNA targets are varied in size significantly since a single mRNA specie can be represented by multiple transcripts and the number of representing transcripts is proportional to mRNA length. It has been demonstrated that random hexamers can overestimate mRNA copy numbers by more than order of magnitude compared with a sequence-specific primer. The oligo(T)s bind to the poly(A) tail and require full-length RNA, which are not an effective choice for transcribing RNA that is likely to be fragmented by partial degradation. Also, the oligo(T) primed reactions usually have significantly lower linear range for reverse transcription and may distort the accurate determination of target abundance.
Recently an alternative approach for quantitation of nucleic acids has been introduced, in which two or more enzymatic reaction produce pyrophosphate (PPi) and generate a detectable luminescence signal (Nyren and Lundin, Anal. Biochem. 1512:504-9.1985; Nyren, Anal. Biochem. 167:235-238, 1987, incorporated herein by reference). The approach is based on detection of released inorganic pyrophosphate during oligonucleotide synthesis by polymerase extension reaction (PCR) as illustrated in Drawing 1. The target nucleic acid molecule can be either RNA or DNA. In series of three consecutive enzymatic reactions the released PPi is converted into ATP by ATP-sulfurylase, and subsequently, the ATP provides the energy to luciferase to oxidize luciferin and generate light. The PPi identification technique is extremely sensitive and potentially can detect a single target DNA or RNA molecule. This is possible because the synthesis of a single large cDNA can require incorporation of thousands of deoxynucleotides, consequently producing thousands of ATP molecules by respective enzymatic reactions and resulting in emission from hundreds to thousands of photons per each cDNA copy synthesized.
Furthermore, for increasing detection sensitivity a bioluminescent regenerative cycle assay system has been recently introduced (Hassibi et al, U.S. Pat. No. 7,141,370, incorporated herein by reference). The regenerative cycle uses the ATP-sulfurylase enzyme (E.C. 2.7.7.4) to convert PPi to ATP. In the presence of luciferin and luciferase the consumption of the ATP molecule results in light emission and formation of a PPi molecule as a by-product. This by-product PPi molecule can be re-used in another cycle of ATP production and subsequent light emission. The advantage of the regenerative bioluminescence system is that each PPi molecule can initiate potentially unlimited number of light emission cycles, producing steady-state emission with the intensity proportional to the number of PPi molecules in sample. Yet, the regenerative system has a serious drawback. The real biological samples unavoidably carry some residual amount of ATP molecules (ATP contamination), that alone with target PPi molecules can be involved as a substrate in the regenerative cycle. In addition, the biological samples exhibit steady-state luminescence (luminescence background), which is originated by non-ATP substrate(s) in samples. The ATP contamination and steady-state luminescence background reduces the sensitivity and distorts the accuracy of PPi measurement by the regenerative cycle assay.
In view of the existing drawbacks the known in the art PPi detection techniques are suitable mostly for qualitative detection in applications such as pyrosequencing, i.e., for determination of DNA sequence (Ronaghi et al., Anal. Biochem. 242:84-89, 1996) and SNP detection (Nyren et al., Anal. Biochem. 244:367-373, 1997, all references herein incorporated by reference in its entirety). Indeed, set alone the interference from contamination and background luminescence, under the existing techniques the bioluminescent signal is proportional to the number of incorporated nucleotides rather than to the number of target molecules. A small number of long oligonucleotide targets can produce comparable luminescence signal as a large number of short oligonucleotides. This drawback makes it difficult or even impossible to measure the number of DNA copies based on the intensity of the bioluminescent signal alone. The same is applicable to the detection of RNA by RNA reverse transcription reaction and bioluminescence detection: the longer RNA sequence is, the more deoxynucleotides are incorporated into cDNA by transcriptase, and the higher the intensity of luminescent signal is detected independently of the actual number of target RNA molecules in sample substance.
Therefore, there are sources of error in PPi detection methods, such as where targets structural differences lead to different efficiencies; or, for example, different replication events are involved for different target sequences; or differences in the rates of probe-target hybridization may exist for different targets thus resulting in varying rates of replications; or random termination of replication reaction; due to the possibility that small differences in replication rate due to non-homogeneous reaction condition result in undesirably large differences in the rate of PPi release when long target molecules are replicated. Therefore, the known in the art methods of enzymatic luminescent detection of nucleic acids in its present state often are not suitable for applications required accurate measurements of the number of copies of target molecules.
While a large number of detection methods for use with nucleic acids and protein arrays have been described in patents and in the scientific literature, virtually all methods set forth in prior art contain one or more inherent weaknesses. Some lack the sensitivity necessary to accomplish certain tasks. Other methods lack the recognition specificity or produce response, which resulted both from the size and the number of target molecules rather than to represent the number of copies of said target molecules. Yet, some methods have drawbacks due to interference from contaminants present in sample. Still others are expensive and difficult to implement or present health safety concerns for workers, who implement these techniques.
Thus, there is a need for an improved method and kit for luminogenic recognition and quantitation of nucleic acid molecules, which said method is quantitative, sensitive, does not required chemical modification of the target molecules for detection and which said method is simple to implement and is able to address drawbacks of the existing techniques. Furthermore, there is a need for providing method, instrumentations, and kits for quantitative comparison of the number of different species of nucleic acid molecules in presence of contaminants producing background luminescence, said method producing luminescent signal proportional to the number of target molecules even if the sequence length of target nucleic acid molecules is varied substantially across different species or even the same specie.
Nomenclature
Unless defined otherwise, all technical and scientific terms used above and throughout the text have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and exemplified suitable methods and materials are described below. For example, methods may be described which comprise more than two steps. In such methods, not all steps may be required to achieve a defined goal and the invention envisions the use of isolated steps to achieve these discrete goals. The disclosures of all publications, patent applications, patents and other references are incorporated in to herein by reference. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Terms that are not otherwise defined herein are used in accordance with their plain, ordinary meaning.
The following definitions are provided to facilitate a clear understanding of the present invention. The term “analyte”, “target”, “target molecule” or “molecular target” refers to a macro-molecule, without limitation as to size, including a nucleoside, nucleotide, oligonucleotide, polynucleotide, nucleic acid, nucleic acid single-stranded or double-stranded polymer and equivalents thereof known in the art, peptide, polypeptide, protein, carbohydrate, polysaccharide, glycoprotein, lipid, hormone, growth factor, cytokine, receptor, antigen, allergen, antibody, substrate, metabolite, cofactor, inhibitor, drug, pharmaceutical, nutrient, toxin, poison, explosive, pesticide, chemical warfare agent, biowarfare agent, biohazardous agent, infectious agent, prion, radioisotope, vitamin, heterocyclic aromatic compound, carcinogen, mutagen, narcotic, amphetamine, barbiturate, hallucinogen, waste product, contaminant, and other biological agents known to those skilled in the art. “Targets” are not limited to single molecules or atoms, but may also comprise complex aggregates, such as a virus, bacterium, Salmonella, Streptococcus, Legionella, E. coli, Giardia, Cryptosporidium, Rickettsia, spore, mold, yeast, algae, amoebae, dinoflagellate, unicellular organism, pathogen or cell. In certain embodiments, cells exhibiting a particular characteristic or disease state, such as a cancer cell, may be targets. Virtually any chemical or biological compound, molecule or aggregate could be a target, so long as it can be attached to a nucleic acid, polynucleotide or oligonucleotide molecule by any type of intermolecular interaction.
The term “nucleic acid” means either DNA, RNA, single-stranded, double-stranded or triple stranded and any chemical modifications thereof. Virtually any modification of the nucleic acid is contemplated by this invention. “Nucleic acid” encompasses, but is not limited to, oligonucleotides and polynucleotides. Within the practice of the present invention, a “nucleic acid” may be of any length.
“Protein” is used herein to refer to any polymer comprised of amino acids, chemically modified amino acids, amino acid analogues and/or amino acid derivatives. The term “protein” encompasses amino acid polymers of any length, from two amino acid residues up to a full length protein. As used herein, the term “protein” encompasses, but is not limited to, peptides, oligopeptides and polypeptides.
The term “bound molecules”, “duplex” or “hybridized molecules” refers to a corresponding pair of molecules held together due to mutual affinity or binding capacity, typically specific or non-specific binding or interaction, including biochemical, physiological, and/or pharmaceutical interactions. Herein binding defines a type of interaction that occurs between pairs of molecules including proteins, nucleic acids, glycoproteins, carbohydrates, hormones and the like. Specific examples include antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, complementary strands of nucleic acid, protein/nucleic acid repressor/inducer, ligand/cell surface receptor, virus/ligand, etc.
The term “sample substance” refers to a media, often a liquid media, which was prepared for the purpose of analysis and establishing (a) the presence or absence of a particular type of molecular target; (b) the presence or absence of a plurality of molecular targets; (c) the presence or absence of specific groups of molecular targets; (d) and if the target molecules are present, to determine/quantify the number of target molecules present in the sample substance.
The term “probe molecular structure” or “probe” refers to a molecule of known nature, which said molecule is capable of binding to a particular type of target molecule or to any biological or chemical agent of interest, or to plurality of target molecules from a specific class/group of molecules. Said probe is used to witness the presence of the corresponding target molecule or a specific class of target molecules in a sample substance.
The abbreviation “DNA/RNA” means “DNA, or equally acceptable, RNA”.
The term “enzymatic luminescence” refers to one or more consecutive biochemical reactions involving at least one reaction of enzyme(s) and substrate(s) which said consecutive reactions are producing light as result of chemical or biochemical modification of the molecules involved.
The term “replication reaction” and “replication of nucleic acid” refers to a enzymatic biochemical reaction involving primer and template, in which reaction the primer and template have a homology and shorter primer molecules is extended according to the sequence of the longer template molecules. Examples of replication reaction include, but not limited to, the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), amplification with Qβ replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35: 117-126 (1991); Landegren, Trends Genetics, 9: 199-202 (1993)) as well as “constant temperature” PCR and rolling circle amplification (Fire A., Xu S. Q, PNAS 92:4641-4645 (1995)), the contents each of which are incorporated herein by reference.
The terms polymerase and reverse transcriptase refers to enzymes known in the art for carrying DNA and RNA replication reactions. Non-limiting examples include DNA polymerases, reverse transcriptases, and RNA-dependent RNA polymerases. Non-limiting examples of polymerases that may be of use include Thermatoga maritima DNA polymerase, AmplitaqFS™ DNA polymerase, Taquenase™ DNA polymerase, ThermoSequenase™, Taq DNA polymerase, Qbeta™ replicase, T4 DNA polymerase, Thermus themophilus DNA polymerase, RNA-dependent RNA polymerase and SP6 RNA polymerase. Commercially available polymerases including Pwo DNA Polymerase from Boehringer Mannheim Biochemicals (Indianapolis, Ind.); Bst Polymerase from Bio-Rad Laboratories (Hercules, Calif.); IsoTherm™ DNA Polymerase from Epicentre Technologies (Madison, Wis.); Moloney Murine Leukemia Virus Reverse Transcriptase, Pfu DNA Polymerase, Avian Myeloblastosis Virus Reverse Transcriptase, Thermus flavus (Tfl) DNA Polymerase and Thermococcus litoralis (Tli) DNA Polymerase from Promega (Madison, Wis.); RAV2 Reverse Transcriptase, HIV-1 Reverse Transcriptase, T7 RNA Polymerase, T3 RNA Polymerase, SP6 RNA Polymerase, RNA Polymerase E. coli, Thermus aquaticus DNA Polymerase, T7 DNA Polymerase +/−3′.fwdarw.5′ exonuclease, Klenow Fragment of DNA Polymerase I, Thermus ‘ubiquitous’ DNA Polymerase, and DNA polymerase I from Amersham Pharmacia Biotech (Piscataway, N.J.), Method of using polymerases and compositions suitable for use in various methods are well known in the art (e.g., U.S. Pat. No. 7,141,370) and incorporated herein by reference in its entirety.
The abbreviation ATP refers to adenosine-5′-triphosphate, CAS No. 56-65-5.
The abbreviation PPi refers to inorganic pyrophosphate, CAS No. 13472-36-1.
The abbreviation APS refers to adenosine 5′-phosphosulfate, CAS No. 485-84-7.
The abbreviation PCR refers to Polymerase Chain Reaction, which is technique for amplifying quantity of DNA, see, e.g., U.S. Pat. Nos. 5,656,493; 5,234,824; and 5,187,083, the contents each of which are incorporated herein by reference.
The abbreviation RT-qBLA refers to real time quantitative PCR method known in the art for detection and quantitation of nucleic acids.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a target molecule” may include a plurality of macro-molecules, including organic compounds, antibodies, antigens, virus particles, metals, metal complexes, ions, cellular metabolites, enzyme inhibitors, receptor ligands, nerve agents, peptides, proteins, fatty acids, steroids, hormones, narcotic agents, synthetic molecules, medications, nucleic acid single-stranded or double-stranded polymers and equivalents thereof known to those skilled in the art, and so forth.